Transcriptional initiation is often controlled by the action of promoter-specific transcription factors acting on the basal transcription apparatus. Typically these factors are soluble proteins that interact with specific components of RNA polymerase. Transcription of several virulence genes in the bacterium Vibrio cholerae is regulated by two membrane-localized activators, ToxR and TcpP, each of which requires specific effector proteins, ToxS and TcpH, which have an unclear function but which are also membrane-localized. The current model for coordinate regulation in this pathogen is that ToxR and TcpP cooperate to activate transcription of the gene encoding another activator, ToxT, which then activates expression of genes encoding cholera toxin, toxin-coregulated pilus and accessory colonization factor. The model also proposes that ToxR, independently of TcpP, simultaneously activates ompU and represses ompT expression. Dr. DiRita proposes genetic and biochemical experiments aimed at analyzing the mechanism of how ToxR and TcpP control gene expression in V. cholerae. The aims of the proposed work are: 1) to determine the molecular basis for transcription activation of toxT by ToxR and TcpP; 2) to determine the significance of ToxR and TcpP-mediated regulation in vivo by reprogramming toxT, ompU and ompT gene expression; 3) to analyze ToxR and TcpP interaction with the transcription machinery to test the hypothesis that these membrane proteins activate transcription directly; 4) to characterize ToxR-dependent activation of ompU; and 5) to study the importance of membrane localization in ToxR and TcpP function. Membrane-localized activators like ToxR and TcpP have been identified in other Gram negative bacteria, including some pathogens. Successful completion of these aims will therefore contribute to a greater mechanistic understanding of a growing family of unusual and important transcription factors.